DESCRIPTION: NAD+ depletion causes neuronal death in rodent models of brain ischemia/reperfusion injury, Wallerian degeneration, multiple sclerosis and occurs after excitotoxic insults and oxidative stress. We have recently discovered that NAD+ depletion is also the primary cause of neuronal death induced by a misfolded and toxic form of the amyloidogenic prion protein (TPrP), showing for the first time the role of NAD+ depletion in the pathogenesis of a protein misfolding neurodegenerative disease (PMND), a family of diseases that comprise, among others, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. We showed that NAD+ replenishment reverses the fate of TPrP-injured degenerating neurons in culture and improves motor function in a mouse model of prion disease. Our hypothesis is that NAD+ replenishment is a novel therapeutic strategy for providing neuroprotective and neurorestorative benefits for many debilitating brain conditions. With TPrP-induced neuronal death as a model, we developed a high throughput screening (HTS) approach to discover small molecules restoring neuronal viability by NAD+ replenishment. The primary and confirmatory assays, optimized in the 384-well plate format, fulfill the criteria of HTS-readiness (Z'>0.5, reproducibility, DMSO tolerance). We have demonstrated the operational robustness of the primary assay using a pilot screen with a small collection of structurally diverse small molecules (LOPAC1280TM). We propose to transfer the assays to the Molecular Libraries Production Centers Network (MLPCN) laboratory at Scripps Florida for miniaturization and screening of a 160K subset of the Scripps Drug Discovery Library. We will then select hits based on the specificity of intracellular NAD+ restoration, EC50/TC50 and activity in cultured primary neurons and in vivo. We will also prioritize hits based upon chemical tractability, potential for selectivity, and drug-likeness. In this medicinal chemistry component of our plan we will also study analogs to improve effects. Mode of action studies will identify cellular targets o selected compounds and provide insights into degenerative pathways linked to NAD+ depletion. Our coordinated multidisciplinary plan addresses all objectives of the NIH program announcement PAR-12-058 (Solicitation of Assays for High Throughput Screening (HTS) to Discover Chemical Probes). Our team will deliver a set of partially optimized, novel, target-specific and tractable small molecules suitable for development into novel lead compounds for neuroprotective therapy in a number of brain conditions. Moreover, these compounds will be widely useful as molecular probes for identifying therapeutic opportunities and for studying the mechanisms of neuronal death in brain conditions linked to a failure of NAD+ metabolism.